Vol. 25, No. 6ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, June 1984, p. 729-7340066-4804/84/060729-06$02.00/0Copyright © 1984, American Society for Microbiology

Pulmonary Deposition and Clearance of Aerosolized InterferonPHILIP R. WYDE,l* SAMUEL Z. WILSON,1 MICHAEL J. KRAMER,2 CHONG-SON SUN,1 AND VERNON

KNIGHT1Department of Microbiology and Immunology, Baylor College of Medicine, Houston, Texas 77030,1 and Department of

Immunotherapy, Hoffmann-La Roche, Inc., Nutley, New Jersey 071102

Received 9 January 1984/Accepted 29 March 1984

Small particle aerosols of a hybrid DNA recombinant human alpha interferon, A/D bgl, and a relatedDNA recombinant leukocyte interferon, A, were generated and delivered to mice for 23.5 h a day for 4consecutive days. The antiviral activity of these interferons in delivery reservoirs, in the aerosols generated,and in the lungs of test mice was monitored during and after aerosol administration in cytopathic effectinhibition assays, using vesicular stomatitis vXirus as the indicator virus. In addition, the activity of theseinterferons in primary mouse embryo cells against influenza AIHK/68 (H3N2) virus was determined. Theresults obtained indicated that the interferon particles generated in the continuous aerosol therapy systemused in these studies remained biologically active and could be readily detected in both aerosol mists andlungs of test mice; levels of exogenous interferon in the lungs equalled or exceeded levels of interferonsproduced endogenously during experimentally induced influenza virus infection. Titers of the exogenouslyadministered interferons decreased gradually and disappeared from the lungs between 24 and 48 h aftercessation of aerosolization. Recombinant human alpha interferon A/D, but not recombinant leukocyte alphainterferon A, significantly inhibited replication of A/HK/68 virus in primary mouse embryo cells in the invitro studies.

Endogenous production of interferons occurs during manyrespiratory virus infections (1, 4, 8, 16, 18, 33, 35) and isthought to be an important factor in recovery from infectionscaused by these viruses (2, 9, 10, 15 17, 32). This view issupported by findings that show the development of moresevere virus disease in T-cell-deprived mice in whom aninterferon response is negligible (17) and in mice treated withanti-mouse interferon globulin in whom the interferon re-sponse is impaired or absent (9, 10, 15) than the diseasewhich occurs in control animals similarly challenged. On thisbasis, many efforts have been made to either stimulateinterferon responses artificially or administer interferonsexogenously as prophylaxis or treatment of several virusinfections (5-7, 14, 25, 26, 28, 31, 32). The results of theseefforts have varied and have not been notably successful,possibly due to differences in susceptibility of different virusstrains to interferon or due to inefficient delivery of thematerial to the site of infection (5, 11, 12, 19). We haveattempted to improve the delivery and effectiveness ofexogenously administered interferon by dispensing this ma-terial in small-particle aerosols. Interferons given in this wayshould deposit throughout the respiratory tract (20).

In the following report, we describe the delivery, deposi-tion, and clearance of exogenously administered interferonsfrom the lungs of test mice given these materials as continu-ous small-particle aerosols over a 4-day period. Highlypurified human alpha interferons produced by using recom-binant DNA technology and affinity columns coated withspecific monoclonal antibodies (27) were used in these testsbecause of their unusually high antiviral activity and avail-ability; moreover, one of the recombinant interferons(rIFNs), hybrid recombinant alpha interferon A/D bgl(rIFNa A/D), has unique activity in mouse tissues (21, 22,29, 34). In addition to the questions concerning delivery,

* Corresponding author.

deposition, and clearance, in vitro studies which test thesensitivity of a mouse-adapted influenza virus to the antiviralactivity of the rIFN were performed. Finally, a comparisonof lung titers of exogenously introduced interferon withlevels of endogenous interferon produced after pulmonaryinoculation with influenza virus was made.

MATERIALS AND METHODSMice. Swiss outbred mice (6 to 9 weeks of age) obtained

from Charles River Breeding Laboratories, Inc., Wilming-ton, Mass., were used in these experiments. These micewere housed in cages covered with barrier filters and werefed mouse chow and water ad libitum.

Viruses. The isolation, adaption to mice, and characteriza-tion of the influenza A/HK/68 (H3N2) (mouse passage 9)virus used in these experiments have been described in detailpreviously (38). Briefly, lungs were homogenized by usingHanks balanced salt solution containing 2% gelatin as adiluent, filtered through a 0.45-,um filter (Acrodisc; no. 4181;Gelman Sciences, Inc., Ann Arbor, Mich.), portioned, andstored at -70°C. Detection and titration of this virus wereroutinely done in Madin Darby canine kidney cells asdescribed previously (39).

Vesicular stomatitis virus (VSV) was obtained initiallyfrom Paul Glezen, Department of Microbiology, BaylorCollege of Medicine, Houston, Tex. Stocks of VSV wereprepared by infecting flasks of L929 fibroblast (L929) cellswith seed virus and harvesting cells and media at the timethat more than 90% cytopathic effect (CPE) was observed.After one freeze-thaw cycle, the resulting suspensions werecentrifuged (100 x g), and the supernatant fluids wereportioned and stored at -70°C. Titration of this virus wasroutinely performed in mouse L929 cells.

Tissue cultures. Starting cultures of MDCK, HEp-2, andL929 cells were obtained from Flow Laboratories, Inc. (no.03-360, 03-108, and 03-439, respectively; McLean, Va.);Madin Darby bovine kidney (MDBK) cells were provided by

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the Department of Experimental and Applied Biology, Hoff-mann-La Roche, Inc., Nutley, N.J. Each of these cellcultures was routinely serially passaged when confluent byusing Eagle minimal essential medium supplemented with 2mM L-glutamine, 100 U of penicillin per ml, 100 p.g ofstreptomycin per ml, 0.2% sodium bicarbonate, and 10%fetal calf serum (10% FCS-MEM). Primary mouse embryo(PME) cells were prepared as described previously (39).Briefly, mouse embryos were cut into small pieces and thencovered with neutral protease (Dispase; grade II; 2.4 U/ml;Boehringer Mannheim Biochemicals, Indianapolis, Ind.).The dissociated cells and remaining fragments were washed,pelleted, and resuspended in 20% FCS-MEM and added to150-cm2 flasks. The resulting monolayers ofPME cells werepassaged when confluent, using 10% FCS-MEM as themedium. Passage levels 2 to 4 of the PME cells were used inthese studies.

Interferons. Both rIFNa A and rIFNa A/D were obtainedfrom the Department of Immunotherapy, Hoffmann-LaRoche, Inc. The construction and specific molecular activi-ties of these interferons have been described in detailpreviously (29). Natural human (no. 12051) and mouse (no.22051) alpha interferons were obtained from Lee Biomolecu-lar Research Laboratories, Inc., San Diego, Calif.Human type alpha (cat. no. G-023-901-527) and mouse

type 1 (no. G-002-904-511) interferon reference reagentswere obtained from June K. Dunnick and John R. LaMon-tagne of the Antiviral Substance Program, National Instituteof Allergy and Infectious Diseases, National Institutes ofHealth (NIH), Bethesda, Md.Measurement of antiviral activity. Assays were carried out

using 96-well round-bottom plates (no. 76-013-05; FlowLaboratories, McLean, Va.). All dilutions and tissue culturesuspensions were prepared with 5% FCS-MEM. Ordinarilyeach assay was performed in triplicate and in parallel in L929and MDBK cells. In some assays, samples were also testedin HEp-2 or PME cells. Briefly, 0.05 ml of medium, testsample, or interferon reference standard was added to thefirst well of each row and serially diluted (twofold), usingmicrotiter loops (Rotatiter; no. 002-961-0100; Dynatech Lab-oratories, Inc., Alexandria, Va.). The appropriate tissuecells were added next in 0.5-ml amounts (ca. 2 x 104 cells perwell), and the plates were incubated at 37°C overnight. Thenext morning, the medium was removed with a suctiondevice, and plates were challenged with either VSV orinfluenza virus (ca. 100 50% tissue culture infective doses in0.1 ml). In assays in which VSV was used, virus controlwells were observed daily. When CPE was 90 to 100%evident in these wells (usually at 48 h), all wells wereobserved, and the degree of CPE in each was recorded.Interferon titers (per 0.05 ml) were expressed as the recipro-cal of the last dilution of each sample which inhibited CPE50% as compared with virus control wells. In assays inwhich influenza virus was used as a challenge virus, themedium in every well was tested on day 5 after the additionof virus for the presence of hemagglutinating virus by usingchicken erythrocytes (0.5% suspension) as indicator cells. Inthis instance, the presence of interferon was indicated by theinhibition of hemagglutination, and interferon titers (per 0.05ml) were expressed as the reciprocal of the last dilutionwhich completely inhibited hemagglutination. Titers of theinterferon reference standards used in these assays rarelyvaried by more than twofold from assay to assay. All titersobtained in L929 and PME cells were adjusted to be relativeto the stated titer of the mouse NIH interferon referencestandard; similarly titers obtained in MDBK and HEp-2 cells

were calculated relative to the NIH human interferon refer-ence standard.

Collection of lungs. Lungs were removed intact withthoracic trachea from mice which were anesthetized withsodium pentobarbitol and bled out by severing the axillaryartery. Each lung was trimmed of detectable lymph nodes,rinsed in sterile saline, and ground in glass homogenizerscontaining 1 ml of5% FCS-MEM. After centrifugation of thehomogenates to remove cellular debris, the resulting super-natants were frozen at -70°C until assayed for interferonlevels.

Aerosol therapy. Aerosol machines containing Collisonnebulizers modeled after a design described by K. R. May(23) were used to generate continuous small-particle aerosolsof interferon. Our use of similar machines to deliver otherantiviral agents has been described in detail previously (36).In these experiments, 6 x 107 U of rIFNa A/D or 1 x 107 Uof rIFNa A were diluted in 350 ml of sterile saline (0.9%sodium chloride; no. 0338-0049-02; Travenol Laboratories,Inc., Deerfield, Ill.) and placed in delivery reservoirs (500-mlpolypropylene beakers (no. 146706; Spectrum Medical In-dustries, Inc., Los Angeles, Calif.). The interferon aerosolswere administered to mice held in plastic cages with sealed-on plastic tops, as described by Young et al. (40). Mice wereexposed to each aerosol for 23.5-h intervals for 4 consecu-tive days. At the end of each 23.5-h interval, the interferonremaining in the reservoirs was checked for titer and micro-bial sterility. In some experiments a control group of micewhich were treated with aerosolized sterile saline not con-taining rIFN was also included.

All-glass impingers were used to collect aerosol samples(3) for assessment of interferon levels in the mists generated.These samples were collected in saline from the ends of themouse cages distal to the aerosol-generating machines with avacuum pump. Duplicate samples were taken for each test,and each sample was of a 2-min duration (25 liters of air).Aerosol samples were also collected by vacuum in anAndersen cascade sampler onto 12% gelatin to determine theaerodynamic mass median diameter (AMMD) of the aerosolparticles generated (23). After collection, the gelatin wasmelted at 37°C, and after thorough mixing, each sample wasserially diluted 1:4 in 5% FCS-MEM. Both the all-glassimpinger and cascade samples were tested for antiviralactivity as described above. In three replicate experiments,the mean AMMD of the interferon particles generated wasestimated to be 2.3 p.m (data not shown). Although particlesizes ranged from 0.6 to 6.6 ,um, more than 90% of theparticles had an AMMD of less than 5.0 p.m.

Statistics. Geometric mean titers (GMTs), standard devi-ations, standard error of the means, and statistical signifi-cance by Student's t test were determined for each sample asdescribed by Sokal and Rohlf (30). All data were trans-formed (log2) before statistical analysis. However, all GMTsare presented as whole numbers except in Fig. 1.

RESULTSAntiviral activity of rIFN in different tissues. Table 1

compares the antiviral activity of rIFNa A and rIFNa A/Dwith natural human and mouse alpha interferons in assays byusing human (HEp-2), mouse (L929 or PME), and bovine(MDBK) tissue culture cell lines and either VSV or influenzaA/HK/68 virus as the challenge virus. As indicated, mousealpha interferon had detectable activity only in mouse L929or PME cells, whereas human alpha interferon and rIFNasignificantly protected HEp-2 and MDBK cells challengedwith VSV and either did not protect or had significantly

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TABLE 1. Comparison of the antiviral activities of rIFNa A andrIFNa A/D with natural human and mouse alpha interferons

Interferon GMT' in:Interferon

tested HEp-2 + L929 + MDBK + PME +VSV VSV VSV A/HK/68

Mouse alpha 0 1,260 0 416Human alpha 630 0 512 0rIFNa A 65,536 32 75,281 0rIFNa A/D 80,684 23,170 75,281 2,048

a Values obtained in mouse L929 and PME tissues were calculated relativeto a mouse reference interferon reagept (NIH no. G4002-902-511); valuesobtained in HEp-2 or MDBK cells were calculated relative to a humanrefere'nce interferon reagent (NIH no. G-023-901-527). In tissues challengedwith VSy, the titer (per 0.05 ml) equals the reciprocal of the last dilutionwhich inhibited VSV-induced CPE .50% as compared with virus controls.Titers in PME cells challenged with influenza A/HK/68 virus equal thereciprocal'of the last dilution of interferon in which no hemagglutinatingactivity was observed. 0, Undetectable activity; number of replicate experi-ments, 3.

reduced antiviral activity in L929 cells challenged with VSVor in PME cells challenged with influenza A/HK/68 virus. Incontrast to all of the other interferons, rIFNa A/D wasalmost equally active in' HEp-2, L929, and MDBK tissuecells and had relatively high GMTs when this material wastested in PME cells, using influenza A/HK/68 virus as thechallenge virus. Although the same interferon reagents wereused in both the VSV and influenza interferon assays, thevalues resulting from the two assays were not comparablesince different tissues, challenge viruses, and endpoints wereused in'the two test systems. Nevertheless, the data (Table1) demonstrate that rIFNa A and rIFNa A/D could bedistinguished from each other and from mouse-derived inter-ferons by their different patterns of antiviral reactivity whentested in parallel in a panel of tissue cell lines derived fromdifferent animal species.

Stability of interferon in generation reservoirs. The antivi-ral titers of the interferon suspensions in the aerosol deliveryreservoirs were monitored at intervals during each aerosolexperiment. As indicated in Table 2, which displays repre-sentative data from these experiments, when rIFNa A/Dwas being aerosolized, significant declines in antiviral titerswere often observed in delivery reservoirs late in each test.(During 16 tests, declines in reservoir titers of rIFN,a A/Dranged from 53 to 96%; declines for rIFNa A generally w'ereless, ranging from 5 to 50%.) The loss in antiviral activity justdescribed was not observed when suspensions of test inter-

TABLE 2. Antiviral activity of rIFNa A/D in aerosol deliveryreservoirs at different intervals during 23.5-h test periodsa

Expt Test Interferon GMT at: %no. tissue 15 min 6 h 19 h 23.5 h Decreasec

1 MDBK 1,123,835 741,455 ND 172,950 85L929 561,918 456,419 ND 122,294 78

2 MDBK 345,901 244,589 244,589 114,105 53L929 244,589 185,363 46,341 80,684 67

'rIFN a A/D was diluted in physiological saline (pH 7.2). Samples wereremoved from the reservoirs at the times indicated and tested in triplicate inMDBK or mouse L929 cells.

b The titer (per 0.05 ml) equals the reciprocal of the test dilution whichinhibited VSV-induced CPE .50% as compared with virus controls. Numberof replicate experiments, 3; ND, not done.

Percent decrease in -mean titer at 23 h as compared with levels observed in15-min samples.

TABLE 3. Antiviral activity of interferon in small-particleaerosols'~~~~~Interferon GMTb in:Expt Interferon tested

no. L929 MDBK

1 rIFNa A 0 162 rIFNa A 0 8

1 rIFNa A/D 119 912 rIFNa A/D 64 39a Titers (per 0.05 ml) of rIFNa A and rIFNot A/D in the reservoirs at the

start of both experiments were similar and, as determined in MDBK cells,averaged 65,536 and 212,927, respectively. Two-minute (25 liters of air)samples were collected by using all-glass impingers containing saline. Only 7-h samples are shown; similar levels were observed at earlier intervals.

b The titer (per 0.05 ml) equals the reciprocal of the last dilution inhibitingVSV-induced CPE 250% compared with virus controls.

ferons were left at room temperature or at 4°C for 23.5 h(data not shown), indicating that the decreases in antiviralactivity observed during aerosolization were related to me-chanical disruption; late in each interval, higher turnoverrates occur because of evaporation and decreased volumesin delivery reservoirs. No turbidity in residual reservoircontents was ever observed in these experiments. More-over, no microbial growth on sheep blood or nutrient agarplates streaked with these residues was ever observed.These data indicate that microbial degradation of the testinterferons was not responsible for the losses in interferontiters noted above.

Activity of interferon in aerosols. The antiviral activity ofthe aerosolized particles collected in all-glass impingers (2-min samples; 25 liters of air) are displayed in Table 3.Although only the results from 7-h samples obtained in twodifferent experiments are shown, values of samples collectedat other times were similar and did not differ significantlyfrom the values depicted (P > 0.05). As indicated, aerosolsof rIFNa A/D in both experiments were active in L929 andMDBK cells. In contrast, samples of aerosols of rIFNa A inthe two experiments exhibited significant antiviral activityonly in MDBK cells. These data clearly indicated that thesmall particles of interferons generated were biologicallyactive.

Levels of rIFNa A and rIFNat A/D in lungs of aerosolizedmice. Table 4 depicts levels of interferon in lungs of mice

TABLE 4. Levels of recombinant alpha interferons in lunghomogenates from mice exposed to continuous small-particle

aerosols of interferonsa

Aerosol Lung interferon GMTb in MDBK cells at (h):content 0 1 3 6 23

Saline 0 0 0 O 0rIFNa A 0 4.6 14 64 64rIFNa A/D 0 16 45 74 91

a Titers (per 0.05 ml) of rIFNa A and IFNa A/D in the delivery reservoirs atthe start of this experiment were approximately 65,536 and 212,927, respec-tively. Lungs from uninfected mice exposed to continuous small particleaerosols for 23.5 h were removed from the mice at the intervals indicated andhomogenized. After centrifugation to remove large debris, the supernataptfluids were tested in MDBK and L929 tissue cells for antiviral activity. Onlylung fluids from mice exposed to rIFNct A/D had activity on L929 cells. Thevalues obtained on L929 cells were essentially the same as the values depictedabove for MDBK cells and thus are not presented.

b The titer (per 0.05 ml) equals the reciprocal of the last dilution whichinhibited VSV-induced CPE 250% as compared with virus controls. 0,Undetectable activity; number of replicate experiments, 3.

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IFNOFF

8

7

6

I-

I-

z0

z

HOURS AFTER VIRUS INOCULATION OR rIFN AEROSOLIZATION

FIG. 1. Interferon levels in lungs of non-virus-inoculated miceexposed to rIFNa A/D administered as a small-particle aerosol(solid line) or in mice inoculated intranasally with influenza A/HK/68 virus but not exposed to aerosols of rIFN (dashed line). GMTs(log2) ± standard deviation are depicted (number of mice per point,4).

exposed to small-particle aerosols of saline, rIFNa A, orrIFNa A/D for 23 h. As indicated, no virus inhibitorysubstances were detected in clarified lung homogenates ofuntreated mice (0 h) or of mice exposed to aerosolizedsaline. In contrast, after only 1 h of aerosolization, signifi-cant levels of interferon were detected in the lungs of miceexposed to aerosols of rIFNa A and in the lungs of miceexposed to aerosols of rIFNa A/D. Levels of interferon inlungs from these mice were still higher at 3 h and reached aplateau by 6 h; no significant increases or decreases in lunginterferon levels were observed between samples obtained at6 and 23 h.

Figure 1 compares levels of rIFNa A/D in the lungs ofnon-virus-inoculated mice who received rIFNa A/D bysmall-particle aerosol for four consecutive 23.5-h intervalswith levels of interferon in the lungs of mice inoculatedintranasally with influenza A/HK/68 virus and not exposedto aerosols of rIFNs. As indicated, endogenously producedinterferon (Fig. 1, dashed line) was detectable in virus-infected mice by 24 h after virus inoculation and reachednear maximum levels 48 h later (GMT log2, 6.5 ± 1.0).Significant declines in lung interferon titers were observed inthese mice at 120 h (GMT log2, 4.0 + 1.0) and again at 144 h(GMT log2, 1.1 + 0.7). Interferon was undetectable in thisgroup at 168 h. In contrast, significant levels of rIFNa A/D(GMT log2, 3.4 + 0.6) were detectable as early as 1 h afterinitiation of aerosolization in the lungs of test mice adminis-tered this interferon exogenously. In this group, interferontiters were near maximum levels by 3 h (GMT log2, 6.0 + 0)and reached a plateau by 24 h (GMT log2, 6.6 ± 0.6).Interferon titers in the lungs of these mice decreased afterthe cessation of aerosolization, but interferon was stilldetectable 24 h after the machines were turned off. Nointerferon was detectable in the lungs of mice from thisgroup 48 h after aerosolization was stopped.

DISCUSSION

The unusual antiviral activity of rIFNa A/D in bothmurine and human tissue cells (21, 22, 29, 34; Table 1),makes this hybrid rIFN an excellent tool to use in mouse-respiratory virus models to begin to evaluate delivery meth-ods, toxicity, immunoregulatory activity, and efficacyagainst respiratory viruses of rIFNs administered exoge-nously. As an initial step in this process, we tested thefeasibility of aerosolizing small particles of rIFNa A/D and arelated recombinant interferon, rIFNa A, which in contrastto rIFNcx A/D, has been shown not to interact readily withinterferon receptor sites on mouse cells (29) or to be veryeffective in protecting mice from lethal virus infection (21,34). The latter interferon serves not only as a second testinterferon but could prove to be an important control infuture studies which involve the determination of whether anobserved effect of rIFNot A/D requires interferon receptorsite interactions.The aerosol route of administration used in these studies

was chosen for several reasons. First, earlier studies byseveral groups of investigators testing the effectiveness ofexogenously administered interferons on respiratory diseaseviruses in humans and animals indicated that parentallyadministered interferons may not reach the lung and nasalmucosa, the primary target areas of respiratory virus infec-tion, in sufficient quantities or for long enough periods oftime to be effective (11, 12, 19). Second, two other antiviraldrugs, amantadine and ribavirin, have been successfullyadministered by continuous small-particle aerosol to humans(19) and mice (37) experiencing influenza infection; in mice,these drugs were shown to be significantly more efficaciouswhen delivered by this method than when given parentally.The results of this study confirmed earlier findings (29)

that rIFNa A/D is highly active on mouse tissue cells in vitroin assays in which VSV is used as a challenge virus (Table 1),whereas rIFNa A, one of the parent interferons from whichrIFNa A/D was derived, is not. In addition, we were able toshow that rIFNa A/D, but not rIFNa A, significantlyinhibited the replication of influenza A/HK/68 virus in PMEtissue (Table 1). The activity that rIFNa A exhibited inmouse L929 cells when tested at high concentrations (Table1) has been described previously and has been attributed tothe forcing of heterologous interferon molecules into tissuecell receptors by mass action (29). Nevertheless, the antivi-ral activity of rIFNa A was more than 2,000-fold less inmouse L929 than in human HEp-2 or bovine MDBK cells(Table 1).Because of their different reactivity patterns in MDBK,

L929, and HEp-2 tissue cells, the two recombinant interfer-ons could be distinguished from each other and from naturalmouse alpha interferon (Table 1). Taking advantage of thisprinciple, we were able to consistently detect and measurelevels of the rIFNs in aerosols and in the lungs of exposedmice. Sera from these animals exhibited low levels ofnonspecific virus inhibition (GMT, 4 to 8), which wasdetectable on all test tissues but only rarely had antiviralactivity attributable to rIFN (based on reactivity patterns).The latter activity was usually low (GMT, 8 to 16) andpresent only in sera tested immediately after aerosolizationswere stopped (data not shown). The marked antiviral activi-ty both in aerosols and in fluids from the lungs derived frommice exposed to aerosols clearly indicated that the testinterferons had biological activity as aerosolized small parti-cles and were being delivered to the lungs in significantquantities. Indeed, significant levels (P < 0.05 versus time 0}

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of rIFNa A/D and rIFNa A were detected in the lungs of testmice after only 1 h of aerosolization, and maximum levels ofinterferons were noted in the lungs of mice by 6 h afterinitiation of delivery (Table 4, Fig. 1). Thus, at the reservoirconcentrations used, interferon titers in the lungs of miceexposed to interferon aerosols equalled or exceeded levels ofendogenous interferon produced in mice during influenza A/HK/68 virus-induced disease, and these levels were reachedwithin hours after aerosol initiation. Such rapid rises ofexogenously administered interferons in lungs suggest thataerosolized interferons may be useful in treating virus-induced respiratory infections, particularly those infectionscaused by viruses which are poor interferon inducers (e.g.,respiratory syncytial virus) or infections in hosts who areimmunologically deficient. Determination of the specificantiviral activity in aerosols of interferon was not deter-mined since protein levels in the aerosol samples collectedwere well below detection limits.rIFNa A and rIFNa A/D titers in delivery reservoirs often

decreased during the 23.5-h aerosolization test periods.Despite these declines, no significant decreases in levels ofinterferon in the lungs of exposed mice were observed duringaerosolization (Table 4, Figure 1). The plateauing of interfer-on levels in the lungs of test mice within 6 h after startingaerosolization and failure of these lung interferon levels torise above plateau values despite 4 days of nearly continuousaerosolization suggested that above physiological levels,exogenously administered interferons were rapidly eliminat-ed from the lungs. In contrast, the continued presence ofexogenous interferon in the lungs of test mice 24 h afterstopping aerosol treatments (Fig. 1) indicated that at physio-logical concentrations, clearance of exogenous interferonswas less rapid. Together, these findings suggest that muchshorter intervals of administration (e.g., 8 h) may lead tolevels of interferon in exposed lungs comparable with levelsobserved in mice exposed for 23.5 h.

It has been determined that particles exceeding an AMMDof 5 pLm deposit primarily in the upper respiratory tract,whereas particles with an AMMD of 3 or less penetratethroughout the respiratory tract (13). By using the Andersoncascade sampler and the procedure of May (24) to estimatesize distribution, it was determined that more than 90% ofthe particles of interferon generated in the aerosol machinesused had an AMMD of less than 5.0 ,um and that the meanAMMD of the particles generated was 2.3 ,Lm. The detectionof signficant levels of exogenous interferon in the lungs fromtest mice confirmed that the administered interferons wereindeed penetrating the lungs.The results in this study indicate that rIFNa A/D is active

on mouse tissues and is effective in vitro against influenza AlHK/68 virus. Moreover, it was demonstrated that by usingCollison nebulizers biologically active small particles ofrIFN can be generated and deposited in the lungs of testmice. Studies to test the effectiveness of this hybrid rIFNgiven by aerosols against influenza and other respiratoryvirus infections in mice are currently underway. In addition,we hope to examine the toxicity, effect of different deliveryregimens, and immunoregulatory activities of rIFNox A/D.

ACKNOWLEDGMENTThis work was supported in large part by a grant from Hoffmann-

La Roche, Inc., Nutley, N.J.

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4. Cate, T. R., R. G. Douglas, Jr., and R. B. Couch. 1969.Interferon and resistance to upper respiratory virus illness.Proc. Soc. Exp. Biol. Med. 131:631-636.

5. Dunnick, J. K., and G. J. Galasso. 1979. Clinical trials withexogenous interferon: summary of a meeting. J. Infect. Dis.139:109-124.

6. Fitter, N. B. 1973. The assay and standardization of interferonand interferon inducers, p. 135-361. In N. B. Finter (ed.),Interferons and interferon inducers, 2nd ed. Elsevier/North-Holland Publishing Co., New York.

7. Greenberg, S. B., M. W. Harmon, P. E. Johnson, and R. B.Couch. 1978. Antiviral activity of intranasally applied humanleukocyte interferon. Antimicrob. Agents Chemother. 14:596-600.

8. Gresser, I., and H. B. Dull. 1964. A virus inhibitor in pharyngealwashings from patients with influenza. Proc. Soc. Exp. Biol.Med. 115:192-195.

9. Gresser, I., M. G. Tovery, C. Maury, and M. Bandu. 1976. Roleof interferon in the pathogenesis of virus diseases in mice asdemonstrated by the use of anti-interferon serum. II. Studieswith Herpes simplex, Moloney sarcoma, vesicular stomatitis,Newcastle disease, and influenza viruses. J. Exp. Med.144:1316-1323.

10. Haller, O., H. Arnheiter, I. Gresser, and J. Lindemann. 1981.Virus-specific interferon action. Protection of newborn Mxcarriers against lethal infection with influenza virus. J. Exp.Med. 154:199-203.

11. Harmon, M. W., S. B. Greenberg, and P. E. Johnson. 1980.Rapid onset of the interferon-induced antiviral state in humannasal epithelial and foreskin fibroblast cells. Proc. Soc. Exp.Biol. Med. 164:146-152.

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